Appreciation of the role of the meninges – a three-layered membrane that covers the brain and the spinal cord and through which the cerebral spinal fluid (CSF) circulates – has expanded from passive physical protection, to dynamic homeostatic functions and serving as a breeding ground of immune activity in inflammatory diseases of the central nervous system (CNS). Yet, despite their newfound recognition as active players in health and disease, composition of the meninges has continued to be evasive and, as a result, full comprehension of their function left disappointingly unrealized. This dilemma has largely been due to an array of technical challenges that restricted compositional analysis of the meninges at the molecular and structural levels. Particularly problematic has been the lack of meningeal protein markers, difficulty in preserving the delicate arrangement of the meninges for microscopic analysis, and inability to view multiple molecular determinants simultaneously. However, new advances in dissecting the meninges, sectioning entire spinal cord with meninges intact, and high-parameter immunohistological image analysis, will allow – for the first time – exhaustive interrogation of the molecular organization of this tissue. This proposal takes advantage of these new developments and proposes a bifurcated approach that will allow us to begin testing the following hypothesis: The composition and/or arrangement of proteins is distinct in the brain vs spinal meninges, and altered during disease. In Aim 1, the individual protein repertoires of meninges from brain and spinal cord of two strains of healthy mice will be revealed using unbiased, label-free quantitative mass spectrometry-based proteomics. Meninges from brain and spinal cord will be evaluated separately, as distinctions in their respective protein components may lie at the basis of why some CNS inflammatory insults strike at particular sites along the neuroaxis. And the respective mouse strains were selected as each will be used, in the next Aim, to model a specific CNS inflammatory condition. In Aim 2, selected proteins identified by proteomics will be targeted by comprehensive immunohistology using Imaging Mass Cytometry (IMC) to characterize the meningeal landscape during health and disease, and its associations with specific leukocyte populations. Pathological conditions will model either the transition from relapsing/remitting-to-secondary progressive multiple sclerosis, or CNS Lupus, as each present with distinctive patterns of CNS inflammation and meningeal involvement. As IMC enables ~ 40 proteins to be localized simultaneously, it offers exceptional ability to elaborate the intricate terrain of the meninges and unveil potential regulatory sites for leukocytes and other immune cells. Such power is still further heightened when IMC is expanded to 3D analysis. Accordingly, by clarifying their molecular makeup, as well as structural alterations and immune interactions of the m...